A creatine and a creatinine are found in blood and urine. A quick and accurate determination of their amounts is very important in making diagnosis of the diseases such as uremia, chronic neophritis, acute neophritis, giantism, tonic muscular dystrophy and the like. For making diagnosis of these diseases, creatine and creatinine in blood, as well as urine are frequently determined quantitatively.
A creatine can be determined by allowing creatine amidinohydrolase and sarcosine oxidase to react on creatine in a sample and determining the amount of the generated hydrogen peroxide by a method for measuring hydrogen peroxide. A creatinine can be determined by allowing creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase to react on creatinine in a sample and determining the generated hydrogen peroxide by a method for measuring hydrogen peroxide.
The creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase are widely found in the world of microorganisms, have been industrially produced and used as reagents for clinical tests.
Yet, the creatine amidinohydrolase produced from various known cell lines show lower heat stability and greater Km value for creatine. For example, an enzyme derived from the bacteria belonging to the genus Bacillus (U.S. Pat. No. 4,420,562) is thermally stable only at a temperature not more than 40° C. An enzyme derived from Pseudomonas putida has a smaller apparent Km value for creatine of 1.33 mM [Archives Biochemistry and Biophysics 177, 508-515 (1976)], though the method for determining the activity is different and the Km value for creatine determined by a coupling assay using sarcosine oxidase and peroxidase widely used as reagents for clinical tests, has been unknown. The enzymes derived from the bacteria belonging to the genus Corynebacterium, Micrococcus, Actinobacillus or Bacillus (Japanese Patent Examined Publication No. 76915/1991) is thermally stable at a temperature not more than 50° C., whereas Km value for creatine is as great as about 20 mM, and these enzymes are not suitable for use as reagents for clinical tests.
In an attempt to resolve such problems, the present inventors previously found that the bacteria belonging to the genus, Alcaligenes produced a creatine amidinohydrolase which was superior in heat stability and had a relatively smaller Km value (Km value: ca. 15.2) for creatine (Japanese Patent Unexamined Publication No. 63363/1994). Furthermore, they have established a technique for isolating a creatine amidinohydrolase gene having a relatively small Km value for creatine from said bacterial cell line and producing said enzyme in a large amount using Gram negative bacteria as a host (Japanese Patent Application No. 117283/1995).
Moreover, a creatine amidinohydrolase stable in a high pH range and having a small Km value has been reported to be derived from the same genus Alcaligenes cell lines (U.S. Pat. No. 5,451,520).
Yet, these creatine amidinohydrolases still have greater Km values as enzymes to be used as routine reagents for clinical tests, and a creatine amidinohydrolase having smaller Km value has been desired.